Q型人血清对氧磷酶在杆状病毒感染的昆虫细胞中的表达

Expression of human serum paraoxonase-1 isoform Q in baculovirus infected insect cells

目的 研究对氧磷酶与心血管疾病的关系。方法 采用BAC TO BAC杆状病毒系统表达Q型人血清对氧磷酶 (PON 1) ,在已获得人PON 1全长cDNA的基础上 ,通过PCR扩增 ,得到上游含EcoRⅠ酶切位点 ,下游不含终止密码子而含HindⅢ酶切位点的PON 1基因 ,将其克隆到带有 6×His·Tag的pFASTBACⅡCD14质粒中 ,构建杆状病毒转移载体pFASTBACⅡ PON 1。将含有PON 1基因的重组质粒pFASTBACⅡ PON 1转化DH10BAC感受态细胞 ,使目的基因通过同源重组插入BacmidDNA中。PCR鉴定正确后再转染入sf9昆虫细胞中 ,产生有感染力的重组杆状病毒。用乙酸苯酯为底物测定表达上清液中的酶活性。结果 比较不同感染复数(MOI)和不同感染时间PON 1在培养上清液中的表达情况 ,确定感染复数MOI为 6～ 8,感染时间 96～10 8h为PON 1的最佳表达条件 ,产率约为 4 .5mg·L- 1上清液。免疫印迹法表明重组蛋白有与抗 6×His单克隆抗体结合的抗原性。顺磁共振实验证明 ,基因工程产人重组血清对氧磷酶rhPON 1能有效清除Fe2 +诱导亚油酸脂质过氧化产生的脂质自由基 ,清除率为 5 5 .9%。结论 BAC TO

AIM To investigate the relationship between paraoxonase and the pathogenesis of cardiovascular diseases. METHODS BAC TO BAC baculovirus expression system to generate human serum paraoxonase(PON 1 isoform Q) was employed. PON 1 gene bearing EcoRⅠ site at upstream and HindⅢ site at downstream was amplified by PCR using the template of the full length PON 1 cDNA, sequenced and then cloned into the plasmid pFASTBACⅡ CD14 with 6× His·Tag to construct the baculovirus transfer vector pFASTBACⅡ PON 1. The recombinant transfer plasmid was transformed into the DH10BAC competent cells, and inserted into the Bacmid DNA by homologous recombination. The sf9 insect cells were infected with the recombinant infective baculovirus. Phenyl acetate was used as substrate to determine the PON 1 activity in the cultural supernatants. RESULTS PON 1 in the cultural supernatants was quantified at different multiplicity of infection(MOI) and time intervals. The best condition for expression was estimated at MOI=6-8, and the infection period of 96-108 h. The overall yield of PON 1 came to 4.5 mg per liter of supernatants. The result of Western blotting showed that the recombinant PON 1 can be recognized by anti 6×His monoclonal antibody. ESR also showed that the recombinant PON 1 could eliminate 55.9% of the lipid free radicals produced during lipid peroxidation of octadecadienoic acid. CONCLUSION The BAC TO BAC baculovirus mediated cells of Spodoptera frugiperda are suitable to express human PON 1 isoform Q in high efficiency.